Protein kinases are ubiquitous enzymes expressed in all eukaryotic cells and are involved in cellular responses to physiological stimuli. Protein kinases attach phosphate groups to substrate proteins. Cyclic-AMP (cAMP) dependent protein kinase (PKA) is an enzyme with broad substrate specificity which phosphorylates substrate proteins in response to cAMP. Protein kinase C (PKC) is an enzyme which phosphorylates substrate proteins in response to intracellular Ca.sup.2+ and phospholipid.
Many hormones act through common signal transduction pathways that generate the intracellular second messenger cAMP. The predominant action of cAMP is to activate a PKA by binding to the regulatory subunit (R) dimer of the holoenzyine thereby releasing the catalytic (C) subunit. Free C subunit potentiates hormonal responses by phosphorylating substrate proteins near the site of kinase activation.
Two classes of the R subunit have been identified; RI and RII subunits which respectively form the type I and type II PKA holoenzymes. The subcellular distributions of PKA isoforms appear to be distinct. The RI isoforms (RI.alpha. and RI.beta.) are reported to be predominantly cytoplasmic and are excluded from the nuclear compartment, whereas up to 75% of the RII isoforms (RII.alpha. or RII.beta.) are particulate and associate with the plasma membrane, cytoskeletal components, secretory granules, the golgi apparatus, centrosomes and/or possibly nuclei. (RII.alpha. or RII.beta.) are particulate and associate with the plasma membrane, cytoskeletal components, secretory granules, the golgi apparatus, centrosomes and/or possibly nuclei.
Intracellular transduction of signals from the plasma membrane to specific subcellular compartments is a complex and highly regulated series of events which control essential physiological processes. An example of signaling pathway involvment are essential in maintaining cellular homeostasis appears in Hunter, Cell, 80:225-236 (1995) where it was shown that many transforming oncogenes encode signal transduction components such as low molecular weight G proteins, protein kinases, or phosphatases. Phosphatases remove phosphate groups from proteins or other compounds. Kinase and phosphatase activities thus control intracellular signal transduction by phosphorylating and dephosphorylating substrate molecules. Now that many genes encoding these proteins have been identified, research emphasis has begun to focus on how these enzymes interface to control cellular events. A critical element in this operation is the subcellular location of each signaling enzyme. For example, Newton, Current Biology, 6:806-809 (1996) showed that the correct intracellular targeting of kinases and phosphatases directs these enzymes to their preferred substrates and reduces indiscriminate background phosphorylation and dephosphorylation.
Kinase and phosphatase targeting is achieved through association with targeting proteins or subunits [reviewed by Faux and Scott, TIBS, 21:312-315 (1996b)]. For example, tyrosine kinase (PTK) and tyrosine phosphatase (PTPase) activity are coupled to downstream cytoplasmic enzymes through adaptor proteins that contain SH2 and SH3 domains. SH2 domains recognize certain phosphotyrosyl residues and SH3 domains bind to a PXXP motif found in some kinases and phosphatases. Modular adaptor proteins like Grb2, p85, IRS-1, Crk and Nck comprise a single SH2 domain that recognizes phosphotyrosyl residues of signalling enzymes and two SH3 domains that bind to the PXXP motif on a separate set of target proteins. Similarly, many phospholipases, kinases, phosphatases and heterotrimeric G-proteins are targeted by specific membrane-targeting motifs such as the LIM, C2, pleckstrin homology and lipid anchoring domains [Gill, Structure, 3:1285-1289 (1995); Newton, Current Biology, 5:973-976 (1995)]. Through these interactions, signaling complexes assemble around receptor kinases or scaffold proteins to mediate cellular processes including hormone signaling events and immune cell function [Harrison et al., TIBS, 20:1213-1221 (1995)].
Until recently, second messenger-stimulated kinases and phosphatases were thought to be localized through association with individual targeting proteins. For example, three classes of phosphatase targeting subunits have been identified which are specific for protein phosphatase I [Chen et al. FEBS Letters, 356:51-55 (1994)]; protein phosphatase 2A [Csortos et al., J. Biol. Chem., 271:2578-2588 (1996)]; or protein phosphatase 2B [Shibasaki et al., Nature, 382:370-373 (1996)]. Likewise, three classes of PKC targeting proteins have been identified in Chapline et al., J. Biol. Chem. 268:6858-6861 (1993); Mochly-Rosen, 1995; and Staudinger et al., J. Cell Biol., 128:263-271 (1995). Compartmentalization of PKA is achieved through interaction of the R subunits with a functionally related family of thirty or so A-Kinase Anchoring Proteins, called AKAPs [reviewed in Scott and McCartney, Molecular Endocrinology, 8:5-13 (1994)]. The present invention contemplates that anchoring proteins confer specificity on serine/threonine kinases and phosphatases by directing these enzymes to discrete subcellular sites where they have restricted access to certain substrates and are optimally positioned to respond to fluctuations in the levels of second messengers.
A variation on this theme was reported in the recent identification of multivalent binding proteins that coordinate the location of serine/threonine kinase and phosphatase signaling complexes. For example, Herskowitz, Cell, 80:187-197 (1995) showed that the pheromone mating response in yeast is initiated through a G-protein linked receptor that activates a yeast MAP kinase cascade. This process proceeds efficiently because each enzyme in the cascade is associated with a scaffold protein called sterile 5 (STE 5) [Choi et al., Cell, 78:499-512, (1994)]. Clustering of successive members in the MAP kinase cascade allows for the tight regulation of the pathway and prevents "cross-talk" between the six functionally distinct MAP kinase modules in yeast [Herskowitz et al., 1995]. Another example of a multivalent binding protein is AKAP79 which targets PKA, PKC and protein phosphatase 2B at the postsynaptic densities of mammalian synapses [Klauck et al., Science, 271:1589-1592 (1996); Coghlan, et al., (1995b). The structure of AKAP79 is modular and resembles STE 5. Deletion analysis, peptide studies and co-precipitation studies of AKAP79 and STE5 have demonstrated that enzymes bind to distinct regions of the anchoring protein [Klauck et al., 1996]. Targeting of the AKAP79 signaling complex to the postsynaptic densities suggests a model for reversible phosphorylation in which the opposing effects of kinase and phosphatase action are co-localized by a common anchoring protein [Coghlan et al., Advances in Protein Phosphatases, 6:51-61 (1995a)].
AKAPs have been identified in a variety of organisms. At least seven proteins that bind the regulatory subunit of PKA in Aplysia californica, a marine invertebrate, have been identified [Cheley et al., J. Biol. Chem., 269:2911-2920 (1994)]. One of these proteins is enriched in crude membrane fractions and taxol-stabilized microtubules and may thus anchor microtubules to the cell membrane as well as bind PKA. A mammalian AKAP microtubule-associated protein 2 (MAP2) attaches PKA to the cytoskeleton [DeCamilli et al., J. Cell Biol., 103:189-203 (1986)]. The PKA-binding site on MAP2 is a 31-residue peptide in the amino-terminal region of the MAP2 molecule [Rubino et al., Neuron, 3:631-638 (1989)].
To date, a number of AKAPs have been identified which apparently bind PKA by a common secondary structure motif that includes an amphipathic helix region [Scott and McCartney, 1994]. Binding of PKA to most, if not all, identified AKAPs can be blocked in the presence of a peptide (Ht31) (SEQ ID NO: 8) that mimics the common secondary structure, while a mutant Ht31peptide containing a single amino acid substitution (SEQ ID NO: 18) that disrupts the secondary structure of the peptide has no effect on PKA/AKAP binding [Carr et al, J. Biol. Chem., 266:14188-14192 (1991)]. Even though PKA/AKAP interaction is effected by a common secondary structure, AKAPs (or homologous AKAPs found in different species) generally have unique primary structure as is evidenced by the growing number of AKAPs that have been identified in a variety of organisms. The unique structure in each anchoring protein confers specificity on a kinase by targeting an AKAP signalling complex to specific intracellular locations.
Chapline and co-workers recently reported the cloning of a PKC binding protein identified as "clone 72" [Chapline et al., J. Biol. Chem., 271:6417-6422 (1996)]. Interestingly, Clone 72 was shown to have substantial homology to a mitogenic regulatory gene identified as "clone 322" [Lin et al., Mol. Cell. Biol., 15:2754-2762 (1995)]. Clone 322 was identified as being the same molecule identified as "SSeCKS" in Lin, et al., J. Biol. Chem. 271:28340-28348 (1996). Clone 322 was shown to be down-regulated in oncogene (e.g., src, ras, fos and myc) transformed cells and thus appears to be a tumor suppressor gene.
Also of interest to the invention is Myasthenia gravis (MG), a disease of neuromuscular transmission characterized by weakness and rapid fatigability of the muscles. It is believed that MG is an autoimmune disease in which the patient develops antibodies to the nicotinic acetylcholine receptor. The nicotinic acetylcholine receptor controls a cation channel in response to binding of acetylcholine. In addition, development of autoantibodies to other cytoskeletal antigens including alpha actinin, actin, filamin and vinculin is observed in the MG patient. The muscle weakness appears to be caused by a failure of the nicotinic acetylcholine receptor as the autoantibodies apparently participate in destruction of the nicotinic acetylcholine receptors.
A previously unknown MG antigen, gravin, was identified by expression screening of a cDNA library with serum from a patient suffering from MG [Gordon et al., J. Clin. Invest., 90:992-999 (1992)]. Gordon, et al. disclosed amino acid sequences disclosing 306 C-terminal amino acid residues of gravin and the corresponding polynucleotide. Gravin was shown to be expressed on the cell cortex and was also shown to be expressed in migratory cells such as fibroblasts and neurons, but not in stationary cells such as epithelial cells. In addition, gravin was found to be expressed in adherent cells, but not in non-adherent cells. Therefore, gravin was postulated to play a role in cell migration and/or cellular adhesion [Grove et al., Anat. Rec., 239:231-242 (1994)].
There continues to exist a need in the art for further insights into the nature, function,and distribution of anchoring proteins and the role of anchoring proteins in myasthenia gravis.